PROJECT SUMMARY/ABSTRACT Despite the high rates of acute graft versus host disease (aGVHD) (up to 50%) and their related mortality/morbidity following allogeneic hematopoietic cell transplantation (allo-HCT), there remains a paucity of therapies and biological correlative studies offered. Current therapies are limited to the nonspecific steroidal targeting of effector cells. Our long-term goal is to identify and validate GVHD biomarkers with the potential for risk stratification and therapeutic targeting. In the previous cycle, we discovered that: (1) soluble STimulation-2 (sST2), the interleukin-33 (IL-33) decoy receptor, as a biomarker for risk of therapy-resistant GVHD and death (N. Engl. J. Med, 2013); (2) Mechanistically, we have shown that during GVHD, sST2 was secreted earlier by intestinal stromal cells and later by cytopathic intestinal T effector cells (Teffs) (Science Translational Medicine, 2015); (3) Furthermore, we have shown that sST2 sequesters IL-33, limiting its availability to T cells expressing the transmembrane molecule form of ST2, mostly cytoprotective regulatory T cells (Tregs) (Science Translational Medicine, 2015; Journal of Clinical Investigation Insights, 2019); (4) Through another proteomics discovery comparing samples at 14 days post-transplantation in patients who develop gastrointestinal (GI) GVHD vs not, we found a T-cell population expressing CD146 that is Th17 prone and ICOS (Inducible T-cell COStimulator)-induced (Journal of Clinical Investigation Insights, 2016). Our new hypotheses address gaps remaining and will be tested with three specific aims: 1) Elucidate the cellular and molecular mechanisms of anti-ST2 neutralizing antibody mediated regulation of inflammation; 2) Implement a prospective multicenter study to determine ST2 threshold as a prognostic biomarker of aGVHD for enabling a biomarker-based preemptive trial; and 3) Inhibit the ICOS/ICOSL pathway with a dual ICOS/CD28 antagonist to prevent and treat aGVHD. The proposed research is significant because the impact of these studies will be 1) to risk stratify patients before initiating GVHD treatment, and 2) to develop entirely novel therapeutic strategies while simultaneously providing novel biological insights into a fatal condition, GVHD.